The rates of hepatic glycogen synthesis in the ADM and control mouse liver in vivo were measured using new advances in13C NMR including 3D localization in conjunction with [1-13C]glucose
Trang 1Elucidation of the role of fructose 2,6-bisphosphate in the regulation
of hepatic carbohydrate metabolism
In-Young Choi1, Chaodong Wu2, David A Okar2, Alex J Lange2and Rolf Gruetter1,3
6
Departments of Radiology1, Biochemistry, Molecular Biology and Biophysics2, Neuroscience3, University of Minnesota Medical School, Minneapolis, MN, USA
Fructose 2,6-bisphosphate (Fru-2,6-P2) plays an important
role in the regulation of major carbohydrate fluxes as both
allosteric activator and inhibitor of target enzymes To
examine the role of Fru-2,6-P2in the regulation of hepatic
carbohydrate metabolism in vivo, Fru-2,6-P2 levels were
elevated in ADM mice with adenovirus-mediated
overex-pression of a double mutant bifunctional enzyme,
6-phos-phofructo-2-kinase/fructose-2,6-bisphosphatase (n¼ 6), in
comparison to normal control mice (control, n¼ 6) The
rates of hepatic glycogen synthesis in the ADM and control
mouse liver in vivo were measured using new advances in13C
NMR including 3D localization in conjunction with
[1-13C]glucose infusion In addition to glycogen C1, the C6
and C2–C5 signals were measured simultaneously for the
first time in vivo, which provide the basis for the estimation of
direct and indirect synthesis of glycogen in the liver The rate
of label incorporation into glycogen C1 was not different
between the control and ADM group, whereas the rate of label incorporation into glycogen C6 signals was in the ADM group 5.6 ± 0.5 lmolÆg)1Æh)1, which was higher than that of the control group of 3.7 ± 0.5 lmolÆg)1Æh)1 (P < 0.02) The rates of net glycogen synthesis, determined
by the glycogen C2–C5 signal changes, were twofold higher
in the ADM group (P¼ 0.04) The results provide direct
in vivoevidence that the effects of elevated Fru-2,6-P2levels
in the liver include increased glycogen storage through indirect synthesis of glycogen These observations provide a key to understanding the mechanisms by which elevated hepatic Fru-2,6-P2levels promote reduced hepatic glucose production and lower blood glucose in diabetes mellitus Keywords:
1 NMR; in vivo; fructose-2, 6-bisphosphate; gly-cogen; mouse liver
The regulation of carbohydrate metabolism in the liver is
important for blood glucose homeostasis by controlling
hepatic glucose production This involves an intricate
regulation of metabolic pathways, such as glycolysis,
gluconeogenesis, glycogenesis and glycogenolysis in the
liver [1,2] The balance of these pathways is severely altered
in patients with type II diabetes mellitus contributing to
chronically elevated plasma glucose concentrations
There-fore, an understanding of the regulation of these fluxes can
provide important insights into the mechanisms and
poten-tial treatment of diabetes
The rates of glycolysis and gluconeogenesis are important
in the rate of hepatic glucose production
Fructose-2,6-bisphosphate (Fru-2,6-P2) plays an important role through
its reciprocal allosteric effects on two critical enzymes,
6-phosphofructo-1-kinase and fructose-1,6-bisphosphatase
(rev iewed in [3]) Fru-2,6-P2 activates
phosphofructo-1-kinase to stimulate glycolysis and inhibits
fructose-1,6-bisphosphatase to reduce gluconeogenesis Synthesis as well
as degradation of Fru-2,6-P2are controlled by the bifunc-tional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bis-phosphatase [4,5], providing a switch between glycolytic and gluconeogenic pathways in the liver [3,6,7] For example, when the insulin/glucagon ratio is high, the enzyme is dephosphorylated at Ser32, its 6-phosphofructo-2-kinase activity is enhanced and the bisphosphatase activity
is inhibited, resulting in a net synthesis of Fru-2,6-P2from fructose 6-phosphate and ATP [5] On the other hand, when the insulin/glucagon ratio is low, 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase is phosphorylated by protein kinase A, which enhances the bisphosphatase activity and inhibits the kinase activity of the bifunctional enzyme, and Fru-2,6-P2 is converted back to fructose 6-phosphate, thereby producing inorganic phosphate (Pi) [3]
Recently, we have shown that increasing Fru-2,6-P2 content via adenovirus mediated 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase overexpression reduces hepatic glucose production and lowers blood glucose in both normal and diabetic mice [8,9] The double mutant bifunctional enzyme used in that study has a mutation of Ser32fiAla, which prevents cAMP-dependent phosphory-lation [10], and a mutation of His258fiAla, which diminishes bisphosphatase activity [11] While the study confirmed that increased hepatic Fru-2,6-P2 content can reduce blood glucose, it was not clear that the allosteric effects of this compound on 6-phosphofructo-1-kinase and fructose-1,6-bisphosphatase could fully account for the metabolic effects, especially with regard to the glycogen
Correspondence to R Gruetter, Center for Magnetic Resonance
Research, 2021 6th Street SE, Minneapolis, MN 55455, USA.
Fax: + 1 612 626 2004, Tel.: + 1 612 625-6582,
E-mail: gruetter@cmrr.umn.edu
Abbreviations: Fru-2,6-P 2 , fructose 2,6-bisphosphate; TR, repetition
time; TE, echo time.
(Received 14 March 2002, revised 10 July 2002,
accepted 18 July 2002)
Trang 2stores In fact, it was observed that the increased hepatic
Fru-2,6-P2 was also correlated with an up-regulation of
glucokinase and a down-regulation of
glucose-6-phospha-tase gene expression, suggesting that this biofactor may also
be involved in balancing the uptake and release of glucose
from the liver [8,9]
13C NMR spectroscopy has been used to measure glucose
and glycogen metabolism in respiring isolated liver cells
[12,13] and perfused liver [12,14–17] Recently, relative flux
rates have been measured in humans [18–21] However,
quantification of absolute fluxes in vivo can be complicated
by the fact that no well-defined three-dimensional
localiza-tion method has been used for hepatic studies and by the
limited amount of information available when measuring
the glycogen C1 signal change alone, resulting only in net
glycogen concentration measurements [22] The present
study presents several advances in the MR technology for
the purpose of measuring hepatic glycogen metabolism
First, this is the first study to implement and use full
three-dimensional localization of13C NMR signals of glycogen in
the intact liver in vivo Second, during infusion of [1-13
C]glu-cose, label incorporation was observed not only into the C1
of glycogen but also the C6, which can only occur by label
scrambling at the level of the trioses In addition, the signals
of glycogen C2–C5 were detected, possibly reflecting
changes in natural abundance glycogen (i.e net glycogen
concentration changes) In the present study, we used these
advances in13C NMR: (a) to measure the rates of hepatic
glycogen synthesis in normal animals treated with
adeno-virus encoding the double mutant rat liver bifunctional
enzyme in comparison with normal (nondiabetic) control
animals; and (b) to assess the role of hepatic Fru-2,6-P2in
controlling glucose and/or glycogen metabolism
M A T E R I A L S A N D M E T H O D S
Animal preparation
The study was conducted according to the guidelines of the
Institutional Animal Care and Use Committee (IACUC) of
the University of Minnesota Twelve male 129J mice
(Jackson Laboratory, Bar Harbor, ME, USA) were studied
after an overnight fast with access to water (n¼ 12,
24.7 ± 0.5 g, mean ± SE) Six normal control animals
were studied without any treatment (control group,
23.3 ± 0.2 g body weight) Six mice were treated with an
adenovirus vector containing the cDNA encoding mutant
rat liver bisphosphatase-deficient 6-phosphofructo-2-kinase/
fructose-2,6-bisphosphatase 7 days prior to the study [8] to
overexpress the double mutant rat liver bifunctional enzyme
(ADM group, 26.0 ± 0.5 g body weight)
All animals were initially anesthetized using a bolus
injection of pentobarbital (Abbott Laboratory, North
Chicago, IL, USA) solution (10 mgÆmL)1) intraperitoneally
(60 mgÆkg)1) Two catheters were inserted into the tail veins,
one for the administration of pentobarbital and one for the
infusion of glucose A third catheter was placed
intraperi-toneally as an alternative means of glucose administration
should the tail vein fail, which was the case in only three
animals, the data of which were included After the lines
were inserted, pentobarbital was infused continuously at
4.8–6.0 mgÆkg)1Æh)1 The animals were secured in a
home-built holder and placed in an acrylic holder attached to an
insert in the gradient coil Body temperature was maintained
at 37.0 ± 0.5C with a warm water circulation system based on a feedback obtained from a temperature probe placed on the abdomen of the mice (Cole Parmer, Vernon Hills, IL, USA) 99% enriched [1-13C]D-glucose (20% w/v solution, Isotec Inc., Miamisburg, OH) was infused either intravenously through the tail vein (n¼ 9) or intraperiton-eally (n¼ 3) with an initial bolus of approximately
50 mgÆkg)1Æmin)1 such that blood glucose was rapidly raised to 10–12 mM Intraperitoneal and intravenous infu-sion protocols were optimized in benchtop experiments to provide a similar rise in plasma glucose in the same short time The glucose infusion rate was adjusted continuously thereafter to maintain a stable liver glucose signal NMR methods
All experiments were performed on a 9.4 Tesla, 31 cm bore horizontal magnet (Magnex Scientific), interfaced to a Varian INOVA console (Palo Alto, CA, USA) An actively shielded gradient coil (Magnex Scientific, Abingdon, UK) with an 11 cm inner diameter was used A custom-built quadrature 1H surface RF coil (14 mm diameter) and a linear three-turn13C coil (12 mm diameter) was used as a transceiver for1H NMR and13C NMR spectroscopy built according to a previously described design [23] A sphere filled with 99%13C enriched formic acid was placed at the center of the13C coil as an external reference and the coil was placed on the animal’s abdomen directly over the liver The position of the liver was identified in gradient-recalled echo magnetic resonance images (repetition time, TR
2 ¼ 10 ms, echo time, TE
interest was placed in the mouse liver with typical volume sizes of 300–430 lL Three-dimensional localization based
on a recently described method [24] that uses outer volume saturation ensured complete elimination of signals from outside of the volume of interest The localized signals of glycogen and glucose were acquired with spectrometer offset set to 100 p.p.m (64 scans with repetition time,
TR¼ 1 s) All data were processed with 15 Hz or 20 Hz exponential multiplication, zero filling, fast Fourier trans-formation
4 and zero-order phase correction.
Glycogen and glucose resonances were quantified using the external reference method as described previously [25,26] In short,13C NMR signals of glycogen and glucose
in vivo were quantified by comparison with the measure-ments of phantoms containing solutions of 400 mM
natural abundance oyster glycogen and 0.9 mM of 99% enriched [1-13C]D-glucose The phantom measurements were performed under identical experimental conditions as the in vivo experiments Coil loading effects on sensitivity and radio frequency (RF)
measur-ing the 180 pulse duration on the13C formic acid signal Differences in saturation factors including T1relaxation and the nuclear Overhauser effect (NOE) were assessed in vivo and in phantom experiments
Assessment of the overexpression of the double mutant bifunctional enzyme
The adenovirus infusion resulted in overexpression of the bifunctional enzyme, which was assessed as described previously [8] The bifunctional enzyme was significantly
Trang 3increased after tail-vein infusion of adenovirus within 3 days
and peaked between 5 and 7 days post infusion The
treatment was accompanied by elevated hepatic Fru-2,6-P2
levels and lowered blood glucose In addition, liver glycogen
content was reduced in response to the overexpression of
ADM relative to the untreated normal control animals [8]
Because a direct assessment of the ADM or Fru-2,6-P2
content in the liver requires tissue extraction, indirect
evidence of ADM overexpression in the liver was assessed
prior to the infusion of glucose from the fasting blood
glucose using a glucose oxidase method (Precision
glucom-eter; Medisense Inc Waltham, MA, USA) and hepatic
glycogen content determined by natural abundance in vivo
13C NMR spectroscopy (see above) This approach is direct
evidence for overexpression of the bifunctional enzyme,
because the protocol was entirely identical to that used in
our previous study [8,9]
Measurement of the rate of13C label incorporation
into glycogen in the liver
Label incorporation into the glycogen C1 reflects glycogen
synthesis via the direct pathway
(glucosefiglucose-6-phos-phatefiglycogen), whereas label incorporation into the
glycogen C6 reflects activity in the indirect pathway due to
label scrambling at the triose level
(glucosefiglucose-6-phosphatefipyruvate (triose level)figlycogen), which is
predominantly a hepatic process Changes in 13C-labeled
glycogen C1 and C6 concentration (rate of 13C label
incorporation, D13Glyc1and D13Glyc6) were calculated by
linear regression at specific time points Data points were
calculated from13C NMR spectra with a temporal
resolu-tion of 4–8 min as a result of averaging spectra collected
with 1 min temporal resolution 13C glycogen changes
were calculated at 2, 3, and > 4 h from the start of
glucose infusion For the calculation of glycogen C6
changes, data acquired within 1 h of the start of the glucose
infusion was not included to avoid any influence of transient
changes in the isotopic enrichment
The rates of 13C incorporation into glycogen were
expressed as a function of precursor metabolite,
glucose-6-phosphate (Glc6P), according to standard tracer
methodo-logy [27]:
d½13Glyc1
dt ðtÞ ¼ Vsyn½
13
Glc6P1
½Glc6P ðtÞ Vphos
½13Glyc1
½Glyc ðtÞ ð1Þ d½13Glyc6
dt ðtÞ ¼ Vsyn½
13
Glc6P6
½Glc6P ðtÞ Vphos
½13Glyc6
½Glyc ðtÞ ð2Þ
Vsyn, Vphos and Vnet represent the flux through glycogen
synthase, phosphorylase, and the rate of net glycogen
synthesis, respectively.13Glyc1 and13Glyc6represent13
C-labeled glycogen C1 and C6 concentration, and13Glc6P1
and13Glc6P6represent13C-labeled glucose-6-phosphate C1
and C6 concentration, respectively Because the rate of label
incorporation into glycogen C1, the relative isotopic
enrichments of glycogen C1 and C6, and the net glycogen
synthesis rate were measured, Eqns (1), (2) and (3) can be
rearranged to determine Vsynand the isotopic enrichment of Glc6P in terms of Vphosas:
½13Glc6P1
½Glc6P ðtÞ ¼
d½ 13Glyc1
dt ðtÞ þ Vphos½13Glyc1
½Glyc ðtÞ
Vnetþ Vphos
ð4Þ
½13Glc6P6
½Glc6P ðtÞ ¼
d½ 13Glyc6
dt ðtÞ þ Vphos½13Glyc6
½Glyc ðtÞ
Vnetþ Vphos
ð5Þ
For example, Vphoscan be determined by measuring 13 C-label dilution during unC-labeled glucose infusion following the13C-labeled glucose infusion, as we have shown recently for brain glycogen [26] Eqns (4) and (5) can be rearranged
to express a relative isotopic enrichment of G6P at the C1 and C6 positions:
½13Glc6P6
½13Glc6P1¼
d½ 13Glyc6
dt ðtÞ þ Vphos½13Glyc6
½Glyc ðtÞ
d½ 13Glyc1
dt ðtÞ þ Vphos½13Glyc1
½Glyc ðtÞ
ð6Þ
Initially, the enrichment of glycogen is low ([13G1yc6]/ [Glyc] << 1) and the temporal changes are approximated
by the slope of the linear regression, which can be used to approximate Eqn (6) as follows:
½13Glc6P6
½13Glc6P1¼
d½ 13Glyc6
dt
d½13Glyc1
dt
ffi½D
13Glyc6
½D13Glyc1
½13Glyc6
½13Glyc1 ð7Þ
Eqn (7) implies that the initial rate of label incorporation into glycogen C6 relative to the rate of label incorporation into glycogen C1 reflects the relative isotopic enrichment of Glc6P in the C6 relative to the C1 position When the changes in glycogen C1 and C6 are linear with time, the differentials in the middle part of Eqn (7) can be replaced by the differences in label incorporation relative to that at time zero (which is close to zero) resulting in the right-hand approximation
R E S U L T S
Localized13C NMR spectra were acquired from the volume
of interest using a three-dimensional localization method The location of the volume of interest, with a nominal volume of 400 lL was based on sagittal and transverse MR images of the mouse liver (Fig 1A) Both the reduced blood glucose (fasting plasma glucose of 5.5 ± 0.3 mMin control
vs 4.1 ± 0.2 mMin ADM mice, mean ± SE) and initial liver glycogen content in the ADM mice were consistent with bifunctional enzyme overexpression in all experiments During infusion of [1-13C]D-glucose, signals from the glucose C1 resonances were immediately detected, along with natural abundance signals from glycerol C1 and C3 at 62.5 p.p.m (Fig 1B, bottom trace) Label incorporation into the glycogen C1 was apparent soon thereafter (Fig 1B, middle trace) followed by label incorporation into a resonance that was clearly resolved from the glycerol C1, C3 resonance This resonance was assigned to the glycogen C6 (Fig 1B, top trace) based on its chemical shift of 61.4 p.p.m [28] and that the linewidth was 77 Hz (after
20 Hz linebroadening), which was clearly broader than the glucose resonances The nearby glucose resonances were not
Trang 4expected to contribute to the glycogen C6 signal change,
because the continuous infusion of [1-13C] glucose will result
in a stable isotopic enrichment for plasma (and liver) glucose
of only a few percentage at C6, leading to a much weaker
signal compared to glycogen C6 The ability to resolve
the resonance of glycerol C1, C3 at 62.5 p.p.m from the
resonance of glycogen C6 allowed, for the first time, the
measurement of glycogen C6 changes in vivo, which reflects
the indirect pathway
13C NMR spectra were acquired while infusing [1-13C]
glucose in an ADM mouse over 7.6 h (Fig 2) The glucose
level in the liver was maintained at 9.7 ± 0.6 lmolÆg)1 (mean ± SE, n ¼ 6, control mice) and at 9.1 ± 0.5 lmolÆg)1(mean ± SE, n¼ 6, ADM mice) throughout the experiments The 13C-label incorporation into hepatic glycogen C1 and C6 increased at a nearly constant rate for the entire measurement period (Fig 2)
In addition to the signal increases in glycogen C1 and C6, increased signals were observed in the spectral region from
70 to 78 p.p.m containing the glycogen C2 through C5 resonances (Fig 2A), which is enlarged in Fig 2B The increase of these signals is consistent with the spectral
Fig 1 1 H MRI and 13 C MRS of the mouse liver (A) Sagittal (left) and transverse (right) images of the liver of a control mouse acquired using the FLASH sequence (TR ¼ 10 ms, TE ¼ 5 ms) The rectangles indicate the location of the volume of interest, 400 lL (8.5 · 8 · 6 mm 3
) The13 C-labeled formic acid sphere can be seen on the left (B) Three-dimensional localized 13 C NMR spectra were acquired from a nominal 400 lL volume
of the control mouse liver during infusion of [1- 13 C] D -glucose The spectra were acquired 0.1 h (bottom), 1.5 h (middle), and 3 h (top) after the start
of the infusion, and each represents an average over 4.3 min (256 scans, 1 s repetition time) Glycogen syntheses via direct and indirect pathways are demonstrated from the increases of signal intensities of glycogen C1 and C6, respectively Data processing consisted of 15 Hz exponential multiplication, zero filling, FFT and zero-order phase correction No baseline correction was applied.
Trang 5Fig 2 Hepatic glycogen synthesis with [1-13C]glucose infusion in an ADM mouse (A) The stack plot of13C spectra acquired over 7.6 h after beginning at t ¼ 0 (right scale), the infusion of [1- 13
C]glucose.13C-label incorporation into hepatic glycogen was detected in the glycogen C1 and C6 resonances Net synthesis of glycogen in the liver is visible from the natural abundance signal increase of glycogen C2–C5 The resonance of glycerol C1 and C3 at 62.5 p.p.m was resolved downfield from the signal of glycogen C6 at 61.4 p.p.m (B) The region containing the glycogen C2–C5 signals (from Fig 2A) was expanded vertically to demonstrate the net synthesis of hepatic glycogen during infusion of glucose Each spectrum corresponds to a 17-min acquisition period Processing consisted of 20 Hz exponential multiplication, with zero filling, FFT and zero-order phase correction The spectra are shown without any baseline correction.
Trang 6pattern of natural abundance glycogen in this region (not
shown) and thus was assigned to reflect primarily increases
in total hepatic glycogen This analysis was based on the
integration of all the C2–C5 glycogen signals To determine
the potential labeling of the glycogen C2 and C5 resonance
due to label scrambling from pyruvate
carboxylase/phos-phoenolpyruvate carboxykinase activity (pyruvate
recyc-ling), the intensity of glycogen C4 was compared to the sum
of the C2, C3, C4 and C5 resonances This comparison
showed that glycogen C4 had the same time course (data
not shown), indicating that pyruvate recycling had a
negligible contribution to the labeling of glycogen under
the conditions of our experiment Natural abundance
signals of glycogen were acquired for 30 min before the
[1-13C] glucose infusion was begun and the quantification
yielded a total glycogen of 246 ± 45.5 lmolÆg)1(mean ±
SE, n¼ 6) in control and 118 ± 27.3 lmolÆg)1(mean ±
SE, n¼ 6) in ADM mice
Time-resolved in vivo measurements of13C-labeled
gly-cogen and glucose in the normal (Fig 3A) and the ADM
(Fig 3B) mouse liver during 4 h of infusion showed that
label incorporation into glycogen C6 lagged compared to
that into glycogen C1 in both groups, consistent with the requirement to reach isotopic equilibrium in the glycolytic intermediates downstream of Glc6P such as at the level of the trioses The ratio of change in glycogen C6 relative to that in glycogen C1 reflects the relative isotopic enrichment
of Glc6P at C6 relativ e to C1 (Eqn 7) At natural abundance, the ratio is one and decreases to a steady-state value after an initial equilibration period This results in a transient change in the Glyc6/Glyc1 ratio, even when the equilibration of the Glc6P pool was instantaneous There-fore, the time required to achieve isotopic steady-state at the Glc6P level was faster than the 0.61 ± 0.05 h required for Glyc6/Glyc1 to approach steady-state (Fig 4) From the ratio of glycogen C6 and C1 (Glyc6/Glyc1), we conclude that the relativ e isotopic enrichment in C6 of Glc6P was significantly higher in ADM than that in control mice The rate of 13C label incorporation into glycogen C1 (synthase flux) was 22 ± 1.5 lmolÆg)1Æh)1(mean ± SE) in the control group and 24 ± 1.8 lmolÆg)1Æh)1in the ADM group, which was not statistically different between the two groups (n¼ 6, p ¼ 0.34, two-tailed t-test, Fig 5) However, the rate of label incorporation into glycogen C6 was significantly lower in the control (3.7 ± 0.5 lmolÆg)1Æh)1, mean ± SE) compared to the ADM group (5.6 ± 0.5 lmolÆg)1Æh)1, mean ± SE) (n¼ 6, p ¼ 0.02) The rate of net glycogen synthesis was significantly lower in the control group (47.2 ± 6.5 lmolÆg)1Æh)1) than the ADM group (95.7 ± 19.9 lmolÆg)1Æh)1) (n¼ 6, p ¼ 0.04) Overall the rates of glycogen metabolism in the control group were lower than those in the ADM group
D I S C U S S I O N
In this study, changes in hepatic carbohydrate metabolism due to the alteration of the activity of bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase were monitored using 3D-localized 13C NMR spectroscopy
Fig 3 Time course of label incorporation into hepatic glycogen and
glucose in control and ADM mice (A) Representative time courses of
glycogen and glucose from the control mouse liver (B) Representative
time courses of glycogen and glucose from the ADM mouse liver The
concentrations of glycogen C1 and glucose C1 resonances are scaled on
the left axis and the concentration of glycogen C6 resonance is scaled
on the right axis The spectra used for this plot were averaged to a
temporal resolution of 8.5 min (512 scans, 1 s repetition time).
Fig 4 Isotopic steady-state at the Glc6P level in the liver in vivo.
13
Glc6P 6 /13Glc6P 1 is approximated by the ratio of the glycogen C6 to the C1 concentration, 13Glyc 6 /13Glyc 1 (Eqn 7); shown is a control mouse (closed circles) and an ADM mouse (open circles) plotted to-gether with the [1-13C]hepatic glucose concentration (top box, with scale to the right) for the control (solid line) and ADM mouse (dashed line) 13 C glucose concentration in the liver was maintained at about
10 lmolÆg)1in both groups as shown in the top plot.
Trang 7Because of its high concentration in hepatic tissue leading to
a high sensitivity, the study focused on the measurement of
the glycogen signals This study represents several novel
advances in in vivo NMR methodology First, the challenges
presented by measuring a well-defined volume of hepatic
tissue in the small liver volume were overcome using a
three-dimensional localization method in conjunction with an RF
coil design optimized for the mouse liver and very high
magnetic field, 9.4 Tesla Although challenges remain in
shimming the signals from the mouse liver, localization was
important to eliminate potential signal sources from
non-hepatic tissue, which was accomplished by a well-defined
volume of interest that concomitantly improved spectral
quality This was evident from the separation of the
glycogen C6 and glycerol C1, C3 signals, an achievement
that, to our knowledge, has not been achieved in the intact
liver in vivo The importance of the detection of label
incorporation into glycogen C6 while infusing [1-13
C]glu-cose can be appreciated from the fact that this labeling
pattern is only possible due to activity of the indirect
pathway of glycogen synthesis In addition, this study
reports for the first time the simultaneous detection of
increased signal intensity for the glycogen C2–C5 resonances
These changes in intensity were attributed to increases in the
natural abundance glycogen concentration, based on the
observation that the rate of the glycogen C4 signal intensity
changed in parallel with the signal intensity of all C2–C5
resonances, and that the spectra shown in Fig 2B closely
resembled those of natural abundance glycogen in aqueous
solutions Therefore, this study reports the first
simulta-neous measurements of label incorporation into glycogen
C1 and C6, as well as changes in total glycogen content
in vivo, which can be used to assess the activity of the direct
and indirect pathway, as well as net glycogen changes
To our knowledge, this is also the first study to apply this
technology to adenovirus transfected mouse liver
Previ-ously, we reported that the levels of Fru-2,6-P were
significantly increased by adenovirus-mediated overexpres-sion of a mutant form of 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase in the mouse liver [8,9] The increased hepatic Fru-2,6-P2 levels resulted in mild hypo-glycemia in normal mice and amelioration of hyperhypo-glycemia
in diabetic animals In this study, the impact of hepatic overexpression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase on glucose and glycogen metabolism due to altered hepatic Fru-2,6-P2levels was monitored using 3D localized13C NMR spectroscopy and the results were consistent with our recent in vitro measurements [8] For example, the liver glycogen concentration determined in vivo
by natural abundance13C NMR spectroscopy just prior to infusion of the [1-13C] glucose was reduced in the ADM mice relative to the control animals The lower hepatic glycogen content in the ADM group reflects increased glycogen utilization in an effort of the liver to establish euglycemia and reflects an overexpression of double mutant 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in the livers of these mice Although this is qualitatively consistent with our earlier results [8], the hepatic glycogen content in this study was approximately 20–25% lower than that determined previously in vitro, which was attributed to the fact that in contrast to the previous work, in the present study mice were fasted for 8–12 h before the NMR experiments were begun Together, these results demon-strate that increased Fru-2,6-P2has similar metabolic effects
in the fed as well as in the fasted state During the NMR experiments, the infusion of [1-13C] glucose was sufficient to maintain blood glucose levels between 10 and 12 mM to induce hyperglycemic states similar to diabetes The glyco-gen repletion observed in both the control and ADM groups was consistent with transition from the fasted to the hyperglycemic (fed) states The striking linearity of the signal increase of glycogen C1 and C6 during this long measurement period (Fig 3) implies measurement of either the early phase of turnover, as the curve did not show any evidence for a leveling of the signal (which suggests a surprisingly long turnover time), or net synthesis of glycogen
or the combination of both
The rates of total glycogen synthesis and 13C label incorporation into glycogen (Fig 5) suggest that the overall rate of glycogen synthesis is much higher than the rate at which the labeled glucose is incorporated into the newly synthesized glycogen This result was unexpected, since the amount of label transferred into glycogen should reflect turnover as well as synthesis and thus be higher than the rate of net glycogen synthesis This observation can be explained, however, by assuming that the isotopic enrichment of Glc6P was much lower than that of glucose A lower enrichment of Glc6P relative to glucose may be due to a slow rate of glucose phosphorylation, or dilution by extra-hepatic precursors and an active indirect pathway of glycogen synthesis Previous work suggests that the latter situation is more likely, as increased hepatic Fru-2,6-P2 was associated with an up-regulation of glucokinase and a down-regulation of Glc6Pase gene expression [8] These observations are also consistent with previous reports suggesting that 30% to 70% of the postprandial liver glycogen is from the indirect pathway [29–31]
Although the increased hepatic Fru-2,6-P2produced by the ADM treatment promoted a higher rate of net glycogen
Fig 5 Rate of glycogen C1, C6 and net glycogen changes during
[1-13C]glucose infusion in the mouse liver in vivo The rates of glycogen
C1 and C6 changes were plotted in the left two pairs of bar graphs (left
axis) and the rate of net glycogen changes was plotted in the right pair
of bar graphs (right axis) The hatched columns are from the control
group (n ¼ 6) and the solid columns are from the ADM group
(n ¼ 6) The rate changes were calculated by linear regression Data
are shown in mean ± SE (error bars) * denotes statistically significant
difference in means based on a two-tailed t-test (P < 0.05, n ¼ 6).
Trang 8synthesis when compared to the normal control mice, the
rate of [1-13C] glucose incorporation was not significantly
increased However, the rate of13C incorporation into the
C6 position in glycogen was significantly increased in the
ADM group, which suggests that the indirect pathway for
glycogen deposition was activated in response to increased
hepatic Fru-2,6-P2 This is a surprising result when
consid-ering that the putative effect of Fru-2,6-P2 is to activate
6-phosphofructo-1-kinase and inhibit
fructose-1,6-bisphos-phatase, which should have reduced the activity of the
indirect pathway of glycogen synthesis, as it depends on flux
through fructose-1,6-bisphosphatase However, it is likely
that the increased rate of13C label incorporation into the C6
position of glycogen in the ADM group is indicative of
activated 6-phosphofructo-1-kinase, which can lead to
increased13C labeling at the triose level Such a mechanism
can lead to increases in labeling of glycogen C6 even in the
presence of decreased activity of the indirect pathway,
provided that the increase in glycolytic flux exceeded the
decreased gluconeogenic flux substantially The ability of
the liver to replenish glycogen stores via the indirect
pathway, even in the face of high Fru-2,6-P2levels, suggests
that the activation of 6-phosphofructo-1-kinase by this
biofactor is more potent than its inhibition of
fructose-1,6-bisphosphatase This is a significant observation because it
offers the first in vivo assessment of the action of Fru-2,6-P2
on the
6-phosphofructo-1-kinase/fructose-1,6-bisphospha-tase cycle The result must be interpreted with care,
however, as both glycolysis and glycogen synthesis have
been shown to be influenced by substrate channeling and
protein–protein interactions [32–34] Such mechanisms may
provide for effective pooling of glycolytic/glycogenic
precursors, i.e Glc6P
In summary, based on several substantial advances in the
13C NMR methodology, the observation of simultaneously
enhanced indirect hepatic glycogen synthesis and glycolysis
in the ADM group has clarified the in vivo action of
Fru-2,6-P2 on the
6-phosphofructo-1-kinase/fructose-1,6-bisphos-phatase cycle, suggesting that the activation of glycolysis
predominates over the inhibition of gluconeogensis These
data, in conjunction with our earlier reports, strongly
suggest that the bifunctional enzyme is an enticing target for
antidiabetic therapies aimed at increasing hepatic
Fru-2,6-P2content
A C K N O W L E D G E M E N T S
This study was supported by the NIH grants R01DK38354 (A J L.)
and two Grants-in-Aid by the University of Minnesota Graduate
School (R G., A J L.) Purchase of 9.4 Tesla magnet was partially
supported by a gift from the W M Keck Foundation and the Center
for MR research is in part supported by a biotechnology research
program grant from the National Center for Research Resources,
P41RR08079.
R E F E R E N C E S
1 Nordlie, R.C., Foster, J.D & Lange, A.J (1999) Regulation of
glucose production by the liver Annu Rev Nutr 19, 379–406.
2 Cherrington, A.D (1999) Banting Lecture 1997 Control of
glu-cose uptake and release by the liver in vivo Diabetes 48, 1198–
1214.
3 Okar, D.A., Lange, A.J., Manzano, A., Navarro-Sabate, A.,
Riera, L & Bartrons, R (2001) PFK-2/FBPase-2: maker and
breaker of the essential biofactor fructose-2,6-bisphosphate Trends Biochem Sci 26, 30–35.
4 Pilkis, S.J., Chrisman, T., Burgress, B., McGrane, M., Colosia, A., Pilkis, J., Claus, T.H & el-Maghrabi, M.R (1983) Rat hepatic 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase: a unique bifunctional enzyme Adv Enzyme Regul 21, 147–173.
5 Van Schaftingen, E & Hers, H.G (1981) Phosphofructokinase 2: the enzyme that forms fructose 2,6-bisphosphate from fructose 6-phosphate and ATP Biochem Biophys Res Commun 101, 1078–1084.
6 Pilkis, S.J., el-Maghrabi, M.R & Claus, T.H (1988) Hormonal regulation of hepatic gluconeogenesis and glycolysis Annu Rev Biochem 57, 755–783.
7 El-Maghrabi, M.R., Pate, T.M., Murray, K.J & Pilkis, S.J (1984) Differential effects of proteolysis and protein modification on the activities of 6-phosphofructo-2-kinase/fructose-2,6-bisphospha-tase J Biol Chem 259, 13096–13103.
8 Wu, C., Okar, D.A., Newgard, C.B & Lange, A.J (2001) Over-expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphospha-tase in mouse liver lowers blood glucose by suppression of hepatic glucose production J Clin Invest 107, 91–98.
9 Wu, C., Okar, D.A., Newgard, C.B & Lange, A.J (2002) Increasing fructose 2,6-bisphosphate overcomes hepatic insulin resistance of type 2 diabetes Am J Physiol Endocrinol Metab.
282, E38–E45.
10 Kurland, I.J., el-Maghrabi, M.R., Correia, J.J & Pilkis, S.J (1992) Rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase Properties of phospho- and dephospho- forms and of two mutants
in which Ser32 has been changed by site-directed mutagenesis.
J Biol Chem 267, 4416–4423.
11 Tauler, A., Lin, K & Pilkis, S.J (1990) Hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase Use of site-directed muta-genesis to evaluate the roles of His-258 and His-392 in catalysis.
J Biol Chem 265, 15617–15622.
12 Cohen, S.M., Ogawa, S & Shulman, R.G (1979) 13C NMR studies of gluconeogenesis in rat liver cells: utilization of labeled glycerol by cells from euthyroid and hyperthyroid rats Proc Natl Acad Sci USA 76, 1603–1609.
13 Cohen, S.M., Rognstad, R., Shulman, R.G & Katz, J (1981)
A comparison of 13C nuclear magnetic resonance and 14C tracer studies of hepatic metabolism J Biol Chem 256, 3428– 3432.
14 Cohen, S.M (1983) Simultaneous13C and31P NMR studies of perfused rat liver Effects of insulin and glucagon and a13C NMR assay of free Mg 2+ J Biol Chem 258, 14294–14308.
15 Iles, R.A., Griffiths, J.R., Stevens, A.N., Gadian, D.G & Porteous, R (1980) Effects of fructose on the energy metabolism and acid-base status of the perfused starved-rat liver A 31phos-phorus nuclear magnetic resonance study Biochem J 192, 191–202.
16 Shulman, G.I., Rothman, D.L., Smith, D., Johnson, C.M., Blair, J.B., Shulman, R.G & DeFronzo, R.A (1985) Mechanism of liv er glycogen repletion in vivo by nuclear magnetic resonance spec-troscopy J Clin Invest 76, 1229–1236.
17 Shulman, G.I., Rossetti, L., Rothman, D.L., Blair, J.B & Smith,
D (1987) Quantitative analysis of glycogen repletion by nuclear magnetic resonance spectroscopy in the conscious rat J Clin Invest 80, 387–393.
18 Rothman, D.L., Magnusson, I., Katz, L.D., Shulman, R.G & Shulman, G.I (1991) Quantitation of hepatic glycogenolysis and gluconeogenesis in fasting humans with13C NMR Science 254, 573–576.
19 Magnusson, I., Rothman, D.L., Jucker, B., Cline, G.W., Shul-man, R.G & ShulShul-man, G.I (1994) Liver glycogen turnover in fed and fasted humans Am J Physiol 266, E796–E803.
20 Roden, M., Perseghin, G., Petersen, K.F., Hwang, J.H., Cline, G.W., Gerow, K., Rothman, D.L & Shulman, G.I (1996) The
Trang 9roles of insulin and glucagon in the regulation of hepatic glycogen
synthesis and turnover in humans J Clin Invest 97, 642–648.
21 Petersen, K.F., Laurent, D., Rothman, D.L., Cline, G.W &
Shulman, G.I (1998) Mechanism by which glucose and insulin
inhibit net hepatic glycogenolysis in humans J Clin Invest 101,
1203–1209.
22 David, M., Petit, W.A., Laughlin, M.R., Shulman, R.G., King,
J.E & Barrett, E.J (1990) Simultaneous synthesis and degradation
of rat liver glycogen An in vivo nuclear magnetic resonance
spectroscopic study J Clin Invest 86, 612–617.
23 Adriany, G & Gruetter, R (1997) A half Volume coil for efficient
proton decoupling in humans at 4 Tesla J Magn Reson 125,
178–184.
24 Choi, I.-Y., Tkac, I & Gruetter, R (2000) Single-shot,
three-dimensional non-echo localization method for in vivo NMR
spectroscopy Magn Reson Med 44, 387–394.
25 Gruetter, R., Ugurbil, K & Seaquist, E.R (1998) Steady-state
cerebral glucose concentrations and transport in the human brain.
J Neurochem 70, 397–408.
26 Choi, I.-Y., Tkac, I., Ugurbil, K & Gruetter, R (1999)
Non-invasive measurements of [1-13C] glycogen concentrations and
metabolism in rat brain in vivo J Neurochem 73, 1300–1308.
27 Watanabe, H & Passonneau, J.V (1973) Factors affecting the
turnover of cerebral glycogen and limit dextrin in vivo J
Neu-rochem 20, 1543–1554.
28 Canioni, P & Quistorff, B (1994) Liver Physiology and Metabo-lism in NMR in Physiology and Biomedicine, pp 373–388 Aca-demic Press, San Diego, CA.
29 Magnusson, I & Shulman, G.I (1991) Pathways of hepatic glycogen synthesis in humans Med Sci Sports Exerc 23, 939–943.
30 Spence, J.T & Koudelka, A.P (1985) Pathway of glycogen synthesis from glucose in hepatocytes maintained in primary cul-ture J Biol Chem 260, 1521–1526.
31 Newgard, C.B., Hirsch, L.J., Foster, D.W & McGarry, J.D (1983) Studies on the mechanism by which exogenous glucose is converted into liver glycogen in the rat A direct or an indirect pathway? J Biol Chem 258, 8046–8052.
32 Yang, R., Cao, L., Gasa, R., Brady, M.J., Sherry, A.D & New-gard, C.B (2002) Glycogen-targeting subunits and glucokinase differentially affect pathways of glycogen metabolism and their regulation in hepatocytes J Biol Chem 277, 1514–1523.
33 O’Doherty, R.M., Jensen, P.B., Anderson, P., Jones, J.G., Ber-man, H.K., Kearney, D & Newgard, C.B (2000) Activation of direct and indirect pathways of glycogen synthesis by hepatic overexpression of protein targeting to glycogen J Clin Invest.
105, 479–488.
34 Agius, L., Centelles, J & Cascante, M (2002) Multiple glucose 6-phosphate pools or channelling of flux in diverse pathways? Biochem Soc Trans 30, 38–43.